Characterization of Thrombate III®, a pasteurized and nanofiltered therapeutic human antithrombin concentrate.

Thrombate III(®) is a highly purified antithrombin concentrate that has been used by clinicians worldwide for more than two decades for the treatment of hereditary antithrombin deficiency. The manufacturing process is based on heparin-affinity chromatography and pasteurization. To modernize the process and to further enhance the pathogen safety profile of the final product, despite the absence of infectious disease transmission, a nanofiltration step was added. The biochemical characterization and pathogen safety evaluation of Thrombate III(®) manufactured using the modernized process are presented. Bioanalytical data demonstrate that the incorporation of nanofiltration has no impact on the antithrombin content, potency, and purity of the product. Scaledown models of the manufacturing process were used to assess virus and prion clearance under manufacturing setpoint conditions. Additionally, robustness of virus clearance was evaluated at or slightly outside the manufacturing operating limits. The results demonstrate that pasteurization inactivated both enveloped and non-enveloped viruses. The addition of nanofiltration substantially increased clearance capacities for both enveloped and non-enveloped viruses by approximately 4-6 log10. In addition, the process achieves 6.0 log10 ID50 prion infectivity clearance. Thus, the introduction of nanofiltration increased the pathogen safety margin of the manufacturing process without impacting the key biochemical characteristics of the product.

Robustness of solvent/detergent treatment of plasma derivatives: a data collection from Plasma Protein Therapeutics Association member companies.

BACKGROUND: Solvent/detergent (S/D) treatment is an established virus inactivation technology that has been applied in the manufacture of medicinal products derived from human plasma for more than 20 years. Data on the inactivation of enveloped viruses by S/D treatment collected from seven Plasma Protein Therapeutics Association member companies demonstrate the robustness, reliability, and efficacy of this virus inactivation method. STUDY DESIGN AND METHODS: The results from 308 studies reflecting production conditions as well as technical variables significantly beyond the product release specification were evaluated for virus inactivation, comprising different combinations of solvent and detergent (tri(n-butyl) phosphate [TNBP]/Tween 80, TNBP/Triton X-100, TNBP/Na-cholate) and different products (Factor [F]VIII, F IX, and intravenous and intramuscular immunoglobulins). RESULTS: Neither product class, process temperature, protein concentration, nor pH value has a significant impact on virus inactivation. A variable that did appear to be critical was the concentration of solvent and detergent. CONCLUSION: The data presented here demonstrate the robustness of virus inactivation by S/D treatment for a broad spectrum of enveloped test viruses and process variables. Our data substantiate the fact that no transmission of viruses such as human immunodeficiency virus, hepatitis B virus, hepatitis C virus, or of other enveloped viruses was reported for licensed plasma derivatives since the introduction of S/D treatment.

The base substitution fidelity of DNA polymerase beta-dependent single nucleotide base excision repair.

Damaged DNA bases are removed from mammalian genomes by base excision repair (BER). Single nucleotide BER requires several enzymatic activities, including DNA polymerase and 5',2'-deoxyribose-5-phosphate lyase. Both activities are intrinsic to four human DNA polymerases whose base substitution error rate during gap-filling DNA synthesis varies by more than 10,000-fold. This suggests that BER fidelity could vary over a wide range in an enzyme dependent manner. To investigate this possibility, here we describe an assay to measure the fidelity of BER reactions reconstituted with purified enzymes. When human uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and DNA ligase 1 replace uracil opposite template A or G, base substitution error rates are